A multiplicity of procedures for isolation of proteins is available and practiced in the art. For soluble proteins such as those that occur in the cytoplasm, direct application of these techniques is possible, as it is unnecessary to take steps to free these proteins from their surroundings. However, proteins or lipids that are membrane-bound, have required harsher treatment to release them from the membrane. One commonly employed technique is solubilization using detergents, or other harsh reagents such as chaotropic agents or other denaturing compounds such as urea. See, for example, Molloy, M. P., et al., Electrophoresis (1998) 19:837-834; Chevallet, M., et al., Electrophoresis (1998) 19:1901-1909. A drawback of such methods is that the isolation process goes too far, i.e., the protein is recovered essentially by itself, and is unaccompanied by the cellular membrane components that normally are associated with it and which are often crucial to its biological function. Elucidation of the biological role of such proteins requires knowledge of the substances involved in transduction of signals resulting from binding of the protein to its specific ligand. Thus, it would be extremely valuable to recover these membrane proteins along with their corollary substances, so that the nature of these substances and the biological role of the protein and its associated substances can be determined. It will be noted that the corollary substances may be associated themselves with the membrane or may be associated with the receptor in the cytoplasm.
Another drawback of the prior art methods is that in some instances, the membranes are sufficiently disrupted by the harsh reagents that cryptic receptors become exposed and coincidentally bind to ligands of interest. Thus, the practitioner is misled into analyzing a receptor with respect to a ligand when in fact the receptor is irrelevant to the biological function of the ligand in question.
A description of one method to obtain lectin receptors from erythrocytes without the use of detergents was described by Jakobovits, A., et al., Biochem. Biophys. Res. Commun. (1981) 100:1484-1490. In this method, erythrocytes were treated with neuraminadase and then with lectin-coated, i.e., peanut agglutinin-coated sepharose beads, thus resulting in coupling the erythrocytes to the beads through an interaction between lectin and the cognate receptors on the cell surface. Unbound cells were removed and the pellet of erythrocyte-covered beads was pipetted vigorously with a Pasteur pipette. The beads were then washed to remove any erythrocytes that had been sheared, and it was determined that most erythrocytes could be removed by repetition of this treatment. Any remaining bound cells could be removed by quick vortexing and subsequent wash. While the erythrocytes were removed, the receptors coupled to lectin remained bound to the sepharose beads. These “plucked” receptors were then released from the beads by boiling or by competitive elution with galactose.
The elution of the bound components resulted in recovery of the lectin binding components designated asialoglycophorins. This work showed that cells lacking nuclei, when treated to expose the relevant sugars, could be bound through carbohydrates on their surfaces to agglutinins (i.e. lectins) and that the carbohydrate-containing moieties bound to the agglutinins could be removed from the cell membranes by mechanical disruption. No description of any non-covalently associated components removed along with the glycoprotein receptors per se was provided. Further, there is no description of cells which had not been treated to expose the relevant sugars, nor was there a suggestion to employ ligands other than lectins. As noted below, lectins are relatively non-specific and thus will bind to a multiplicity of glycosylated proteins some of whose biological functions are irrelevant to the specific nature of the lectin. While effective binding of cells can more readily be achieved through the use of lectins than through the specific ligands of the present invention, the resultant does not provide information regarding biologically relevant receptors due to the non-specificity of lectin binding. Some lectins do engender biological responses, and lectins are specific with respect to the carbohydrates to which they bind, but the individual carbohydrate targets are promiscuous with respect to the receptors in which they reside, thus, as regards individual receptors, specificity of binding is lost.